This invention relates to an improved immunoassay method applying a lysing action of complement for liposome membranes.
Immunoassay is a measuring method utilizing an antigen-antibody reaction. It is widely used as a method for specifically measuring trace components, for example, living components such as proteins, hormones, active peptides, autacoid, tumor maskers, immunoglobulin, etc., drugs such as digoxin, phenytoin, phenobarbital, etc., in body fluids.
As immunoassays which are now generally used, there are radioimmunoassay (RIA), enzyme immunoassay (EIA), etc. These methods permit quantitative measurement of trace components in samples but involve individual problems. That is, RIA is disadvantageous, for example, in that since radioisotopes should be used therein, RIA requires special facilities and troublesome disposal of wastes. EIA is disadvantageous, for example, in that it requires a relatively long measuring time and is difficult to apply to an autoanalyzer.
Therefore, as an immunoassay involving none of these problems, there has recently been proposed and noted an immunoassay using liposomes (hereinafter referred to as "liposome immunoassay"). A typical example of this method is disclosed in Japanese Patent Unexamined Publication No. 56-132564 (U.S. Pat. No. 4,342,826). This method comprises mixing liposomes, surfaces of which are fixed with an analyte to be measured and which encapsulate a marker (e.g. enzyme) therein, a sample and an antibody to an analyte to carry out the antigen-antibody reaction and adding complement thereto. Thus, complement activated by an antigen-antibody complex formed on the surfaces of liposomes, lyses liposome membranes to liberate the marker encapsulated. This liposome immunoassay using complement (hereinafter referred to as "complement immunoassay") does not include the above-mentioned problems of RIA and EIA and can conduct a series of reactions in a uniform reaction system, so that this method is noticed for carrying out the measurement simply and in a short time.
But according to the complement immunoassay, when a series of complement components and a complement controlling factor (ex. C1INE, C3BINA (Factor I), B1H (Factor H), etc.) are present in samples, the liposome lysing reaction by activation of complement is influenced so as to lose the correlation between the amount of an analyte in a sample and the amount of a marker released from liposomes. Thus, when serum or plasma is used as a sample, the released amount of marker is changed by complement activity by the influence of the above-mentioned interfering substance for the measurement contained in such a sample, resulting in failing to obtain precise measured values. In order to remove such an influence in the case of using serum or plasma as a sample, it is recommended to subject the sample to pretreatment, for example, heating at 56.degree. C. for 30 minutes, or at 60.degree. C. for 3 minutes. [Japanese Patent Unexamined Publication No. 1-214762; Gradwhol's Chemical Laboratory Methods and Diagnosis, vol 2, 7th Ed, San Frankel et al., p. 1478 (1970)]. But such a method requires complicated procedures and a longer time, in addition to inconvenience of vaporization of the sample by the heat. Further, it is difficult to apply such a heat treatment method to auto-analyzers now widely used for detection. Further, there is also proposed a method for adjusting the ionic strength of a buffer for reaction for the same purpose (Japanese Patent Unexamined Publication No. 1-214763). But according to this method, when a salt concentration becomes too high, there is a fear of influencing the antigen-antibody reaction.